Biochemical and Structural Characterization of the S. Pombe La-Related Protein 1 (Slr1p)

Abstract

La and La-related proteins (LARPs) are conserved RNA-binding proteins that share a characteristic La motif (LaM) and have important functions in RNA metabolism. Several LARP families exist where members possess specific domains that allow for binding to unique substrates. The LARP1 family, which includes a DM15 domain, binds a cohort of mRNAs encoding factors involved in the process of mRNA translation. These mRNAs contain a sequence of 5-15 pyrimidines in their 5′UTRs, immediately following the m7G cap, and are characterized as 5′ terminal oligopyrimidine (5′TOP) mRNAs. The DM15 domain of human LARP1 has been suggested to specifically recognize this motif, thereby affecting the translation and stability of 5′TOP mRNAs. However, the specific function of LARP1 in this context remains unclear. Intriguingly, the 5′TOP motif does not exist in yeast, and LARP1 orthologs in this system lack the characteristic DM15 domain. In this work, we characterize the S. pombe La-related protein 1, Slr1p, as an RNA-binding protein with conserved functions relative to other LARP1 orthologs. Through proteomic and transcriptomic studies, we show that Slr1p regulates the translation and stability of mRNAs that encode ribosomal proteins and other components of the translational apparatus in S. pombe. We further identify a 5′ Proximal AC-rich (5′PAC) motif, residing close to the start AUG, in these mRNAs and propose that they represent a cohort of proto-5′TOP mRNAs. Through structural analyses and mutagenesis studies, we show that the LaM of Slr1p is responsible for binding to the 5′PAC RNA motif, where mutagenesis of a single amino acid in our characterized binding site disrupts this interaction. We further demonstrate that Slr1p plays a role in adaptation to translation stress and that its phosphorylation status may allow for several functional states. Our findings are consistent with the conservation of LARP1 function across eukaryotes, and suggest that the involvement of this protein family in ribosomal protein mRNA translation and stability is influenced by a more fundamental LaM-dependent form of LARP1 regulation that is utilized in S. pombe.